Sensor membranes

ABSTRACT

The present invention relates to a novel method for producing an electrode membrane combination. This novel method employs compounds including a linker lipid for use in attaching a membrane including a plurality of ionophores to an electrode and providing a space between the membrane and the electrode, the electrode being either in part or totally made up of the linker lipid. The linker lipid comprises within the same molecule a hydrophobic region capable of spanning the membrane, an attachment group used to attach the molecule to an electrode surface, a hydrophilic region intermediate said hydrophobic region and the attachment group and a polar head group region attached to the hydrophobic region at a site remote from the hydrophilic region.

This is a division of application Ser. No. 08/406,853, filed asPCT/AU93/00509, Oct. 1, 1993, published as WO94/07593, Apr. 14, 1994,now U.S. Pat. No. 5,637,201.

FIELD OF THE INVENTION

The present invention relates to electrode membrane combinations for usein ion selective electrodes and biosensors. In addition, the presentinvention relates to methods for the production of such electrodemembrane combinations and the use of ion selective electrodes andbiosensors incorporating such electrode membrane combinations in thedetection of analytes. The present invention also relates to novelcompounds used in the electrode membrane combinations.

BACKGROUND OF THE INVENTION

Lipid bilayer membranes (also known as black lipid membranes--BLM's) arewell known in the biological and chemical fields. The ability ofionophores to modulate the ion flux through these membranes is also wellknown. Modulation of the ion flux of the membrane in response tospecific molecules is also known, especially in the biochemical fields.The lipid bilayer membranes are however extremely fragile and sensitiveto non-specific physical and chemical interference. The preparation andproperties of the BLM's are fully described in textbooks and literaturearticles.

It has been known since 1967 that ionophores incorporate into lipidbilayers (P. Mueller et al, Biochem. Biophys. Res Commun., 26 (1967)298; A. A. Lev et al Tsitologiya, 9 (1967) 102;) in BLM's and that theselective ion flux through the membrane could thus be monitored.Possibility of producing a lipid bilayer containing ionophores on anionic hydrogel reservoir and using such as an ion selective electrodehas also been suggested (U. J. Krull et al, U.S. Pat. No. 4,661,235,Apr. 28, 1987), however no means of obtaining reproducible and stablebilayer membranes have been taught in the art. Using a Langmuir-Blogettbilayer and multilayer approach (T. L. Fare et al Powder Technology, 3,(1991), 51-62; A. Gilardoni et al, Colloids and Surfaces, 68, (1992),235-242) has been attempted however the ion selectivity was inadequateand the response time was too slow for practical purposes, stability wasnot adequate and the LB technique is generally considered to be toodifficult for industrial applications.

Ionophores in the context of the present invention are any of thenaturally occurring lipophilic bilayer membrane compatible ion carrierssuch as valinomycin, nonactin, methyl monensin or other naturallyoccurring ion carriers, or synthetic ionophores such as lipophiliccoronands, cryptands or podands, or low molecular weight (<5000 g/mol)naturally occurring or synthetic ion channels such as gramicidin,alamethicin, mellitin or their derivatives. Additionally trialkylatedamines or carboxylic acids such as phytic acid may serve as protonionophores.

Ion channels may also include large, lipid membrane compatible, proteinion channels, especially where their function and stability is enhancedthrough their incorporation into lipid bilayers that are essentiallyfree of extraneous alkane material.

In the broad context of the present invention lipids are deemed to beany amphiphilic molecules, either naturally occurring or synthetic,containing a hydrophobic hydrocarbon group and a hydrophilic head group.

Biosensors and ion selective electrodes incorporating gated ionophoresin lipid membrane combinations have been disclosed in InternationalPatent Application Nos PCT/AU88/00273, PCT/AU89/00352, PCT/AU90/00025and PCT/AU92/00132. The disclosure of each of these references isincorporated herein by reference.

As is disclosed in these applications, suitably modified receptormolecules may be caused to co-disperse with amphiphilic molecules andproduce membranes with altered surface binding properties, which areuseful in the production of biosensor receptor surfaces of high bindingability and high binding specificities. It is also disclosed thationophores such as polypeptide ionophores may be co-dispersed withamphiphilic molecules, thereby forming membranes with altered propertiesin relation to the permeability of ions. There is also disclosure ofvarious methods of gating these ion channels such that in response tothe binding of an analyte the conductivity of the membrane is altered.The applications also disclose methods of producing membranes withimproved stability and ion flux using chemisorbed arrays of amphiphilicmolecules attached to an electrode surface and means of producing lipidmembranes incorporating ionophores on said chemisorbed amphiphilicmolecules. Additionally, means of co-dispersing ion selective ionophoreswith amphiphilic molecules thereby producing ion selective membranecombinations are disclosed.

The present inventors have now determined improved means of increasingthe stability and ion flux properties of the lipid membranes through theuse of novel synthetic lipids and lipid combinations, and novel means ofmembrane assembly.

In various embodiments the present invention consists in the use ofnovel bilayer membrane spanning lipids and bilayer lipids and methods ofassembly thereof, in order to modulate the properties of the lipidsensor membrane so as to control the ion transport properties of theionophore, the thickness and fluidity of the membrane, the stability ofthe membrane, the response to serum, plasma or blood, and thenon-specific absorption of proteins to the membrane.

SUMMARY OF THE INVENTION

In a first aspect, the present invention consists in a linker lipid foruse in attaching a membrane including a plurality of ionophores to anelectrode and providing a space between the membrane and the electrodein which the membrane is either in part or totally made up of the linkerlipid, the linker lipid comprising within the same molecule ahydrophobic region capable of spanning the membrane, an attachment groupused to attach the molecule to an electrode surface, a hydrophilicregion intermediate said hydrophobic region and the attachment group,and a polar head group region attached to the hydrophobic region at asite remote from the hydrophilic region.

In a preferred embodiment of the present invention, the head groupregion is selected from the group consisting of groups normallyassociated with naturally occurring or synthetic lipids such asglycerol, phosphatidyl choline, phosphatidyl ethanolamine, mono-, di- ortri-methylated phosphatidyl ethanolamine, phosphatidic acid,phosphatidyl serine, phosphatidyl glycerol, phosphatidyl inositol,disubstituted head groups as found in cardiolipins, ganglioside headgroups, sphingomyelin head groups, plasmalogen head groups, glycosyl,galactosyl, digalactosyl, sulfosugar, phosphosugar, N-acetyl neuramicacid, sialic acid, aminosugar head groups, carbohydrate head groups,gal(beta1-3)galNAc(beta1-4) NAcNeu(alpha2-3!gal(beta1-4)glc-ceramide,oligomers of ethylene glycol, ethylene glycol, oligomers of propyleneglycol, propylene glycol, amino acids, oligomers of amino acids,combinations of oligomers of ethylene glycol or propylene glycofunctionalised with amino acids or other ionic species or anycombination or derivative of the above.

It is generally preferred that the head group is a naturally occurringor synthetic head group that can be used to minimise the non-specificbinding of proteins onto the surface of the membrane.

In a further preferred embodiment of the present invention, in order toprovide surface characteristics that minimise the non-specific bindingof proteins, it is preferred that the head group is a polyethyleneglycol ranging in molecular weight of between 600-6000 g/mol.

In a further preferred embodiment, the head group is a phosphatidylcholine group.

In a further preferred embodiment, the head group is a glycerol headgroup.

In a further preferred embodiment, the head group is a biotin or abiotinylated 6-aminocaproic acid group or an N-biotinylated oligomer of6-aminocaproic acid.

In a further preferred embodiment, the head group is aGal(beta1-3)galNAc(beta1-4) NAcNeu(alpha2-3!gal(beta1-4-Glc-ceramidehead group.

In a further preferred embodiment, of the present invention it ispreferred that the head group is a group capable of being used tocovalently link a protein molecule onto the linker lipid. The proteinmolecule may be either a receptor such as an antibody or an antibodyfragment or may be an enzyme or may be a protein molecule chosen inorder to impart biocompatible properties to the membrane.

It is further preferred that the head group is terminated in acarboxylic acid group capable of being used to conjugate the linkerlipid with a protein molecule via the amine groups on the protein.

It is further preferred that the head group is a polyethylene glycol inthe molecular weight range 400-1000 g/mol terminated in a carboxylicacid group.

In a further preferred embodiment, the head group is a group capable ofbeing covalently linked with a protein molecule via the aldehyde groupsgenerated from the oxidation of carbohydrate groups on the proteinmolecule.

In a further preferred embodiment, the head group is a hydrazidederivative.

In a further preferred embodiment, the head group is a polyethyleneglycol terminated in a carboxy hydrazide derivative.

In a further preferred embodiment, the head group is a group capable ofbeing covalently linked to a protein molecule via free thiol groups onthe protein molecule.

It is further preferred that the head group is a maleimide derivative.

In a further preferred embodiment, the head group is a group capable ofbeing covalently coupled to a carboxylic acid group on a proteinmolecule.

It is preferred that the hydrophobic group has the general structure asshown in FIG. 1 where the group (X) is a hydrocarbon chain that isapproximately half the length of the group (Y).

It is preferred that the group (X) will generally be between 10-22carbons in length and may be a saturated, unsaturated or polyunsaturatedhydrocarbon, or may be an alkyl substituted hydrocarbon such as thephytanyl group or other mono- or permethylated hydrocarbon chain.

It is further preferred that the group (X) is a phytanyl group.

In a preferred embodiment of the present invention, the group (Y) inFIG. 1 is a single chain hydrocarbon group of length between 20-60 Ålong.

In a further preferred embodiment the group (Y) consists in a singlechain group that is between 20-60 Å long and contains within the chain arigid spacer group such as biphenyl ether or biphenylamine or otherbiphenyl compound. The rigid spacer group serves the function of makingthe synthesis of the group simpler as it easily enables the coupling oftwo smaller alkyl chains onto the rigid spacer group, enabling longsections of the group (Y) to be synthesised readily. The rigid spacergroup also enhances the ability of the linker lipid to assume themembrane spanning conformation of the linker lipid as opposed to anU-shaped conformation within the membrane.

In a further preferred embodiment, the group (Y) is a single chain groupthat is between 30-50 Å long and contains within the chain a N,N'-alkylsubstituted 4,4'-biphenyl amine group.

In a further preferred embodiment, the group (Y) is a single chain groupthat is between 30-50 Å long and contains within the chain a4,4'-biphenyl ether group.

In a further preferred embodiment, the group (Y) is a bis-hexadecyl4,4'-biphenyl ether.

In a further preferred embodiment, the group (Y) is a bis-tetradecyl4,4'-biphenyl ether.

In a further preferred embodiment, the group (Y) is a bis-dodecyl4,4'-biphenyl ether.

In a further preferred embodiment, the group (Y) is a single chain groupthat is between 20-60 Å long and contains within the chain an alkylsubstituted amine.

In a further preferred embodiment, the group (Y) consist in a singlechain group that is between 20-60 Å long and contains within the chain abis-alkylated pentaerythritol group.

In a further preferred embodiment of the present invention, the membranespanning lipid is a single chain lipid in which the group (X) in FIG. 1is absent.

In a further preferred embodiment, the group (Y) contains groups thatcan alter their conformation in response to an external stimulus such aslight, pH, redox chemistry or electric field. The change in conformationwithin the group (Y) will allow the properties of the membrane such asthickness to be controlled through such external stimulus. This can inturn be used to modulate the conduction of ion channels throughmodulation of the on/off times of the channels and the diffusion of thechannels.

In a preferred embodiment, where the group (Y) alters its conformationin response to light stimulus, the group (Y) contains a 4,4'- or3,3'-disubstituted azobenzene.

In a further preferred embodiment the group (Y) contains a group thatundergoes a spiropyran-merocyanine equilibrium in response to lightstimulus.

In a further preferred embodiment of the present invention, thehydrophobic region of the linker lipid consists of oligomers of longchain amino acids, such as 11-aminoundecanoic acid, 16-aminohexadecanoicacid or other amino acid where the carbon chain is preferably between6-20 carbons long, and where the amino acids are linked via amidelinkages.

It is further preferred that the amide groups are tertiary, alkylsubstituted amide groups, where the alkyl groups are phytanyl groups orsaturated or unsaturated alkyl groups between 1-18 carbons in length.

The nature of the hydrophilic group, the attachment group and theelectrode are as described in PCT/AU92/00132.

As is set out in this earlier application it is preferred that theattachment region of the linker lipid is attached to the electrodesurface by chemisorption. In a situation where the electrode is formedof a transition metal such as gold, platinum, palladium or silver, it ispreferred that the attachment region includes thiol, disulphide,sulphide, thione, xanthate, phosphine or isonitrile groups.

In further preferred embodiment the electrode is formed of gold, silver,platinum or palladium and the attachment region includes either a thiolor a disulfide group, the linker lipid being attached to the electrodeby chemisorption.

In an alternate embodiment where the electrode is formed such that ahydroxylated surface is formed on the electrode, it is preferred thatthe attachment region includes silyl groups such as silyl-alkoxy orsilyl chloride groups. The hydroxylated electrode surface may be aprepared by a number of techniques known to someone skilled in the artand may consist of oxidised silicon or oxidised metals such as tin,platinum, iridium.

In yet a further preferred embodiment the electrode is formed ofoxidized silicon, tin, platinum or iridium and the attachment regionincludes silyl groups, the linker lipid being attached to the electrodeby covalent attachment.

The hydrophilic region of the linker lipid is preferably a long chainhydrophilic compound. The hydrophilic region of the linker lipid may becomposed of oligo/poly ethers, oligo/poly peptides, oligo/poly amides,oligo/poly amines, oligo/poly esters, oligo/poly saccharides, polyols,multiple charged groups (positive and/or negative), electroactivespecies or combinations thereof. The main requirement of the hydrophilicregion of the linker lipid is that it allows the diffusion of ionsthrough the ionophores provided in the membrane. This is achieved by theplacement of suitable ion and/or water binding sites along or within thelength of the long chain that makes up the reservoir region.

In a preferred embodiment of the invention the hydrophilic regionconsists of an oligoethylene oxide group. The oligoethylene oxide groupmay consist of four to twenty ethylene oxide units.

In a further preferred embodiment the hydrophilic region consists of asubunit of tetraethylene glycol attached to succinic acid. Thistetraethylene glycol/succinic acid subunit may be repeated 1-4 times.

In a further preferred embodiment the hydrophobic region of a proportionof the linker lipids have covalently attached thereto an ionophore via ahydrophobic spacer.

As set out above the molecule having a hydrophobic region as shown inFIG. 1 incorporating a rigid spacer group provides a number ofadvantages. This hydrophobic region can, of course, be synthesizedseparately from the hydrophilic region, attachment region, and polarhead group region. This hydrophobic region, or synthetic lipid, isbelieved to be new in its own right and can be included in bilayermembranes as a membrane spanning lipid to improve variouscharacteristics of the membrane, such as stability.

Accordingly, in a second aspect the present invention consists in asynthetic lipid for use in bilayer membranes, the synthetic lipid havinga structure as shown in FIG. 1 in which Y is a single chain group thatis between 20 and 60 Å long and contains a rigid spacer group and X arehydrocarbon chains approximately half the length of Y or are absent.

It is preferred that the group (X) will generally be between 10-22carbons in length and may be a saturated, unsaturated or polyunsaturatedhydrocarbon, or may be an alkyl substituted hydrocarbon such as thephytanyl group or other mono- or permethylated hydrocarbon chain.

It is further preferred that the group (X) is a phytanyl group.

In a further preferred embodiment the rigid spacer group is a biphenylether or biphenylamine or other biphenyl compound.

In a further preferred embodiment, the group (Y) is a single chain groupthat is between 30-50 Å long and contains within the chain a N,N'-alkylsubstituted 4,4'-biphenyl amine group.

In a further preferred embodiment, the group (Y) is a single chain groupthat is between 30-50 Å long and contains within the chain a4,4'-biphenyl ether group.

In a further preferred embodiment, the group (Y) is a bis-hexadecyl4,4'-biphenyl ether.

In a further preferred embodiment, the group (Y) is a bis-tetradecyl4,4'-biphenyl ether.

In a further preferred embodiment, the group (Y) is a bis-dodecyl4,4'-biphenyl ether.

In a further preferred embodiment, the group (Y) is a single chain groupthat is between 20-60 Å long and contains within the chain an alkylsubstituted amine.

In a further preferred embodiment, the group (Y) consist in a singlechain group that is between 20-60 Å long and contains within the chain abis-alkylated pentaerythritol group.

In a further preferred embodiment of the present invention X in FIG. 1is absent.

In a further preferred embodiment the synthetic lipid includes a headgroup. Preferred head groups are those listed in the first aspect of thepresent invention.

The present inventors have determined that the linker lipids describedin the first aspect of the present invention as well as the linkermolecules described in PCT/AU92/00132, where the attachment group is athiol and the hydrophobic region is a single hydrocarbon chain, or wherethe attachment group is a thiol or disulfide group and the hydrophobicregion is made up of two hydrocarbon chains, then, when these linkerlipids are adsorbed onto a freshly prepared noble metal electrode aclose packed monolayer membrane is formed that does not permit theionophore to easily penetrate into the membrane, hence restricting theion flux through the membrane. If electrode surfaces are used that arecontaminated then the adventitious introduction of defect sites, wherethe chemisorption of the sulfur containing groups does not occur, willallow ionophores to penetrate into the monolayer. Contamination of thesurface of a gold electrode can occur by adsorption of contaminants fromair over a period of minutes to hours and results in electrode surfacesthat can suffer from poor reproducibility and stability. The presentinventors have devised a more controlled method of producing membraneswith the required spacing between the linker molecules, while stillretaining the efficient and reproducible attachment of the hydrophilicmolecules onto the electrode surface during the deposition of the firstlayer.

In the prior art it has always been believed necessary that on forming asecond lipid layer onto coated electrodes an apolar containment vesselis required in order to obtain sealed bilayer membranes on solidsubstrates. Additionally, the prior art teaches that an alkaneco-solvent such as decane, dodecane, tetradecane or hexadecane isbeneficial in the formation of highly insulating lipid membranes. Thepresent inventors have now determined means whereby it is possible toform insulating lipid bilayer membranes with a minimal amount or noalkane co-solvent and without the need for an apolar containment vessel.The inventors believe this to be beneficial for control of non-specificserum effects, stability, ion conduction and possibly to reducenon-specific binding of analyte molecules to the containment vessel.Incorporation of ionophores into the bilayer membrane allows the ionflux through the membrane to be modulated depending on the nature of theionophore as taught in the prior art. Additionally, the presentinventors have determined means whereby it is possible to reduce theinterference caused by the presence of serum or plasma on the lipidmembrane by using lipid combinations that reduce the effect ofnon-specific ionophore gating effects.

Accordingly, in a third aspect, the present invention consists in amethod of producing an electrode membrane combination comprising thesteps of:

(1) Forming a solution containing reservoir lipids comprising within thesame molecule an attachment region, a hydrophilic region, a hydrophobicregions, and optionally a head group; and spacer compounds comprisingwithin the same molecule a hydrophilic group and an attachment group;

(2) contacting the electrode with the solution from step (1), thecomposition of the electrode and the attachment regions being selectedsuch that the attachment regions chemisorb to the electrode;

(3) rinsing the electrode;

(4) contacting the coated electrode from step (3) with a solution oflipid and ionophore in a carrier solvent containing less than 2% of analkane such as decane, dodecane, tetradecane or hexadecane; and

(5) adding an aqueous solution to the electrode from step (4).

The hydrophobic region of the reservoir lipid may be either half or fullmembrane spanning.

In a preferred embodiment, the reservoir lipid is23-(20'-oxo-19'-oxaeicosa-(Z)-9'-ene)-70-phenyl-20,25,28,42,45-pentaoxo-24aza-19,29,32,35,38,41,46,47,52,55-decaoxa-58,59-dithioahexaconta-(Z)-9-eneas described in PCT/AU92/00132, referred to hereafter as "linker A" orreservoir phytanyl lipid (B) as shown in FIG. 7 or reservoir phytanyllipid (C) as shown in FIG. 8.

In a preferred embodiment, the spacer molecule is a low molecular weightmolecule containing within the same structure a thio or disulfide groupand one or more hydroxyl or carboxylic acid groups.

In a preferred embodiment, the spacer molecule isbis(2-hydroxyethyl)disulfide or 2-mercaptoethanol.

In a preferred embodiment, the solution of step 1 contains a mixture oflinker A, membrane spanning reservoir lipids andbis-(2-hydroxyethyl)disulfide.

In a preferred embodiment, the solution of step 1 contains a mixture oflinker A, membrane spanning reservoir lipids andbis-(2-hydroxyethyl)disulfide in a ratio of 2:1:3.

In a preferred embodiment, the spacer molecule is mercaptoacetic acid,the disulfide of mercapto acetic acid, mercaptopropionic acid or thedisulfide of mercaptopropionic acid, 3-mercapto-1,2-propanedio or thedisulfide of 3-mercapto-1,2-propanediol.

In a further preferred embodiment the hydrophobic region of a proportionof the reservoir lipids have covalently attached thereto an ionophorevia a hydrophobic spacer.

In a preferred embodiment of the present invention, the lipid andionophore solution contains no alkane such as decane, dodecane,tetradecane or hexadecane.

In a further preferred embodiment the reservoir lipid includes a headgroup. Preferred head groups are those listed in the first aspect of thepresent invention.

In a further preferred embodiment of the present invention the lipid instep (4) is glycerol monophytanyl ether.

In a preferred embodiment of the present invention, the lipid is amixture of glycerol monophytanyl ether and a lipid having a polyethyleneglycol group of between 600-6000 g/mol as a head group.

In a further preferred embodiment, the lipid is a mixture of glycerolmonophytanyl ether and 1-3% of a lipid having a polyethylene glycol headgroup. It is further preferred that the polyethylene glycol has amolecular weight in the range of 600-3000 g/mol.

In a further preferred embodiment, the polyethylene glycol containinglipid comprises in the same molecule a phytanyl group attached to asuccinate group at one end and a polyethylene glycol 2000 attached tothe other end of the succinate.

In a further preferred embodiment, the lipid is a mixture of glycerolmonophytanyl ether and a lipid having a phosphatidyl choline head group.

In a further preferred embodiment, the lipid is a mixture of glycerolmonophytanyl ether and up to 20% of a lipid having a phosphatidylcholine head group.

In a further preferred embodiment, the lipid is a mixture of glycerolmonophytanyl ether and up to 20% of a lipid having a phosphatidylcholine head group and up to 3% of a lipid having as a head group apolyethylene glycol of molecular weight between 600-3000 g/mol.

In a further preferred embodiment, the lipid is a mixture of glycerolmonophytanyl ether and a lipid having a head group in which the headgroup is a Gal(beta1-3)galNAc(beta1-4)NAcNeu)alpha2-3!gal(beta1-4)Glc-ceramide carbohydrate head group.

In yet a further preferred embodiment a plurality of ionophores arefunctionalised with a derivative of a low molecular weight analyte whosepresence is to be detected.

In this arrangement, the addition of an antibody or other receptormolecule will modulate the ion transport properties of the ionophore.This conductance modulation can then be detected using establishedimpedance spectroscopy or other methods and could also be used todirectly monitor the presence of antibodies or other receptors to thelow molecular weight analyte. Addition of the test solution to theelectrode membrane combination containing the ionophore/antibody orreceptor complex will lead to competitive binding of the analyte to theantibody or receptor, thereby allowing the ionophore to again diffusefreely through the membrane. The difference can be determined usingtechniques such as impedance spectroscopy and can be used to determinethe concentration of the analyte in the test solution.

In the alternative arrangement where a synthetic or low molecular weightreceptor is attached to the ionophore, the transport properties of theionophore will be directly modulated on complexation of the analyte ofinterest by the synthetic or low molecular weight receptor molecule.

In yet a further preferred embodiment of the present invention theionophore is capable of transporting an ion that is produced by thereaction of an enzyme with its substrate, said enzyme being eithercovalently or non-covalently attached to the membrane surface.

Addition of a solution containing the substrate will cause the enzyme toproduce ionic species which will be transported by the ionophore acrossthe membrane, thus modulating the conductance properties of themembrane, which will be related to the amount of substrate present insolution.

In a preferred embodiment of the present invention, the enzyme is aurease producing ammonium ions from the substrate urea.

In the case where the enzyme is covalently attached to the membrane, itis preferred that the covalent attachment is via a proportion of thelipid molecules that are suitably functionalised for covalent attachmentto proteins or by covalent attachment to the head group of the reservoirlipid including a head group.

Although the nature of the carrier solvent does not appear critical itis preferred that the carrier solvent is a solvent or solvent mixturewherein the lipid and ionophore is soluble and which is preferably watersoluble. Suitable solvents are common water miscible solvents such asethanol, dioxane, methanol or mixtures of these solvents. Addition ofsmall amounts of non-water insoluble solvents such as dichloromethanemay also be included in the carrier solvent in order to solubilise thelipid and ionophore.

In any of the preferred embodiments of the third aspect of the presentinvention, the containment vessel may be a containment vessel with polarsides.

It is further preferred that the containment vessel is made of materialwith a hydrophilic surface.

It is further preferred that the containment vessel is made of amaterial that minimises non-specific binding of proteins or has itssurface modified in order to minimise protein adsorption on addition ofthe analyte sample to be tested.

In the situation where the reservoir lipid contains within the samemolecule a an unsymmetrical disulfide group, where one of the sulfuratoms has a relatively small organic group attached to it, as theattachment region and a single hydrocarbon chain as the hydrophobicgroup, the cross-sectional area of the disulfide group is larger thanthat of the single hydrocarbon chain, hence a membrane with close packedhydrocarbon groups does not form on adsorption of the reservoir lipid,thus allowing penetration of ionophores into the adsorbed layer.Accordingly in such a situation it is not essential to use spacermolecules.

As described in the third aspect of the present invention, it ispossible to produce membrane layers that are impermeable towardsionophores. The present inventors have determined means whereby it ispossible to produce membrane layers by chemisorption of suitable linkerlipids, including lipid, membrane spanning and ion channel containinglinker molecules, onto an electrode surface such that the conformationof the ion channel and the lipids is controlled. The present inventorshave also determined that the presence of the membrane spanning linkerlipids enhances the stability of the subsequent lipid bilayer formed, aswell as controlling the thickness of the subsequent bilayer membrane. Itis known in the art that the lifetime and hence conduction of gramicidinion channels is controlled in part by the thickness of the bilayermembrane. Thicker bilayer membranes shorten the lifetimes of the ionchannel, thinner bilayers increase channel lifetimes. Hence, it ispossible to control the lifetime of the ion channel by controlling thethickness of the bilayer through the use of membrane spanning lipidsthat have different lengths.

The present inventors have also determined that the inclusion ofmembrane spanning lipid linker lipids that contain polar or bulky headgroups decreases the amount of multilayer structure that is obtained inthe absence of these lipids.

Furthermore, the inventors have determined that the presence of amolecule having a lipid or hydrophobic component in the solvent isbeneficial in enabling the ion channel to assume the proper conformationon adsorption on the electrode surface. Prior art membranes includingion channels are typically formed by doping the formed membrane with ionchannels. It is now believed that such a technique results in a numberof the ion channels being incorporated into the membrane in states whichare non-conducting. Where the channels are helical peptides this may bedue to unravelling of the helix, intermeshing of a number of the ionchannels to form non-conducting helices, or that the longitudinal axisthrough the helical peptide is substantially parallel to the plane ofthe membrane and is thus unable to facilitate the transport of ionsacross the membrane. The present inventors have developed a method ofattaching such ion channels to an electrode such that a higherproportion of the channels exist in a conducting form. The membranecombination thus formed contains a mixture of half-membrane spanningreservoir lipids, membrane spanning reservoir lipids and conducting ionchannel linker compounds formed in a close packed layer such thatnon-linker ion channels do not penetrate into the first layer when thesecond layer is formed on the coated electrode.

In a fourth aspect the present invention consists in a method ofproducing an electrode membrane combination, the method comprising thefollowing sequential steps:

(1) Forming a solution comprising ion channels having attached at an endthereof a reservoir region, the reservoir region including a hydrophilicgroup and an attachment group; and a reservoir lipid, the reservoirlipid comprising a hydrophobic region, a hydrophilic region, anattachment region and optionally a head group in a polar carriersolvent;

(2) Contacting the electrode with the solution from step (1), thecomposition of the electrode and the attachment groups and theattachment regions being selected such that the attachment groups andthe attachment regions chemisorb to the electrode;

(3) After a period of incubation rinsing the coated electrode from step(2) to remove unbound material;

(4) Adding to the rinsed electrode from step (3) a solution comprisingion channels and a lipid in a carrier solvent; and

(5) Adding to the electrode from step (4) an aqueous solution such thata lipid bilayer membrane coating the electrode is formed.

In a preferred embodiment of the present invention the ion channels aregramicidin or analogues or derivatives thereof.

In a further preferred embodiment, the gramicidin is a functionalisedgramicidin that consists in the same structure of a gramicidin backbone,an hydrophilic group and a disulfide group as shown in FIG. 13 and willbe referred to hereafter as "linker gramicidin B".

In a further preferred embodiment the reservoir lipid is reservoir lipidA(23-(20'-Oxo-19'-oxaeicosa-(Z)-9'-ene)-70-phenyl-20,25,28,42,45,-pentaoxo-24-aza-19,29,32,35,38,41,46,47,52,55-decaoxa-58,59-dithiahexaconta-(Z)-9-ene)or reservoir phytanyl lipid (B) or reservoir phytanyl lipid (C).

The hydrophobic region of the reservoir lipid may be either half or fullmembrane spanning.

In a further preferred embodiment the reservoir lipid includes a headgroup. Preferred head groups are those listed in the first aspect of thepresent invention.

In a further preferred embodiment the reservoir lipid has a biotincontaining head group.

In a further preferred embodiment, the reservoir lipid has a head groupused to couple the reservoir lipid to a protein molecule or otherreceptor molecule.

In a further preferred embodiment, the reservoir lipid used in step 1has a head group used to minimise the non-specific serum interaction onthe membrane such as a polyethylene glycol or phosphatidyl cholinegroup.

In a further preferred embodiment, the solution in step 1 furtherincludes a lipid. The lipid in the solutions in step 1 and step 4 may bethe same or different.

In a further preferred embodiment, the lipid used in the polar solventin the solution of step 1 is any natural or synthetic lipid that allowsgramicidin to assume the correct conformation for deposition onto theelectrode surface in a conducting state, in the lipid solvent mixtureused.

In a further preferred embodiment, the lipid in step 1 used in the polarsolvent is a glycerol monoalkenoate.

In a further preferred embodiment, the lipid is glycerol monooleate.

In a further preferred embodiment, the polar solvent is ethanol,methanol, trifluoroethanol.

In a further preferred embodiment, the solvent is ethanol or methanol.

It is generally preferred that the electrode from step 2 is treated withan aqueous solution prior to step 3. This is preferably done as soon aspossible after contacting the electrode (step 2) with the solutionformed in step 1. This, however, is not essential. For example where thepolar solvent in step 1 is methanol there is no need to add an aqueoussolution to the coated electrode from step 2.

The aqueous solution used in this optional step and in step 5 may be ofa large number of solutions such as 0.1 to 1.0M saline.

In a further preferred embodiment, the electrode is treated with asolution prepared in step 1 where the polar solvent is methanol, andwhere the electrode is rinsed in step with a suitable solvent such asethanol or methanol.

In a further preferred embodiment of the present invention, the solutionin step 1 comprises 140 mM glycerol monooleate, 1 mM reservoir phytanyllipid (B), and 0.0014 mM linker gramicidin B in ethanol or methanol.

In a further preferred embodiment, the solution in step 1 comprises 140mM glycerol monooleate, 1.4 mM reservoir phytanyl lipid (B), 0.0014 mMlinker gramicidin B, and 0.0014 mM membrane spanning linker lipids inethanol or methanol.

In a further preferred embodiment, the solution in step 1 comprises 140mM glycerol monooleate, 14 mM reservoir phytanyl lipid (B), 0.0014 mMlinker gramicidin B, and 0.014 mM membrane spanning linker lipids inethanol or methanol.

In a further preferred embodiment, the linker gramicidin B concentrationis varied from 0.000014 mM to 0.014 mM.

In a preferred embodiment the membrane spanning phytanyl lipid (B)concentration is varied from 0.1 mM to 1 mM.

In a preferred embodiment the membrane spanning linker lipidconcentration is varied from 0.0001 mM to 1 mM.

In a preferred embodiment of the present invention, the lipid is aglycerol monoalkenoate where the alkenoate group may be an unsaturatedhydrocarbon chain of between 16-22 carbons in length.

In a further preferred embodiment, the lipid is a glycerol monoalkylether where the alkyl group is a hydrocarbon chain of between 16-22carbons in length and may contain unsaturation or may be substitutedwith methyl groups in order to lower its phase transition.

In a preferred embodiment of the present invention, the lipid isglycerol monooleate, glycerol monopalmitoleic, mono-11-eicosenoin,mono-erucin.

In a further preferred embodiment, the lipid is glycerol monooleate ormono-11-eicosenoin.

In a preferred aspect of the present invention, the lipid is glycerolmonophytanyl ether.

In a further preferred embodiment of the present invention, the lipidconsists in a mixture of glycerol monoalkyl ether or glycerolmonoalkenoate and a lipid where the head group consists in apolyethylene glycol group of between 600-6000 g/mol.

In a further preferred embodiment, the lipid is a mixture of glycerolmonoalkyl ether or glycerol monoalkenoate and 1-3% of a lipid where thehead group is a polyethylene glycol group. It is further preferred thatthe polyethylene glycol has a molecular weight in the range of 600-3000g/mol.

In a further preferred embodiment, the polyethylene glycol containinglipid comprises in the same molecule a phytanyl group attached to asuccinate group at one end and a polyethylene glycol 2000 attached tothe other end of the succinate.

In a further preferred embodiment, the lipid is a mixture of glycerolmonoalkyl ether or glycerol monoalkenoate and a lipid where the headgroup is a phosphatidyl choline head group.

In a further preferred embodiment, the lipid is a mixture of glycerolmonoalkyl ether or glycerol monoalkenoate and up to 20% of a lipid wherethe head group is a phosphatidyl choline head group.

In a further preferred embodiment, the lipid is a mixture of glycerolmonoalkyl ether or glycerol monoalkenoate and up to 20% of a lipid wherethe head group is a phosphatidyl choline head group and up to 3% of alipid where the head group is a polyethylene glycol of molecular weightbetween 600-3000 g/mol.

In a further preferred embodiment, the lipid is a mixture of glycerolmonoalkyl ether or glycerol monoalkenoate and a lipid where the headgroup is a gal(beta1-3)galNAc(beta1-4) AcNeu(alpha2-3)! gal(beta1-4)AcNeu(alpha2-3)! carbohydrate head group(Galβ1-3-GalNAcβ1-4Gal(3-2α-NeuAc)β1-4Glc-1-1 ceramide head group).

Although the nature of the carrier solvent does not appear critical, itis preferred that the carrier solvent is a solvent or solvent mixturewherein the lipid and ionophore is soluble and which is preferably watersoluble. Suitable solvents are common water miscible solvents such asethanol, dioxane, methanol or mixtures of these solvents. Addition ofsmall amounts of non-water insoluble solvents such as dichloromethanemay also be included in the carrier solvent in order to solubilise thelipid and ionophore.

In any of the preferred embodiments of the fourth aspect of the presentinvention, the containment vessel may be a containment vessel with polarsides.

It is further preferred that the containment vessel is made of amaterial with a hydrophilic surface.

It is further preferred that the containment vessel is made of amaterial that minimises non-specific binding of proteins or has itssurface modified in order to minimise protein adsorption on addition ofthe analyte sample to be tested.

In yet a further preferred embodiment of this aspect of the presentinvention the solutions in steps (1) and (4) contain less than 2%, andpreferably 0% of an alkane such as decane, dodecane, tertradecane orhexadecane.

Without wishing to be bound by scientific theory it is believed that themethod of the present invention provide a greater proportion ofconducting ion channels due to the fact that the ion channels in step 1are able to assume their native configuration in the lipid component ofthe reservoir lipid. These ion channels are then laid down and attachedto the electrode via the attached reservoir regions in thisconformation. The rinsing of the membrane to remove the unboundreservoir lipid then removes all unbound ion channels. This shouldresult in the majority of the bound ion channels being in a conductiveconfiguration. The subsequent addition of a lipid results in theformation of a lipid bilayer with the bound ion channels present in thelower layer. The ion channels added in step 4 will then partitionprimarily in the upper layer.

Another advantage provided by the method of this aspect of the presentinvention is that it enables accurate adjustment of the proportion ofion channels in the upper layer of the membrane bilayer.

The present inventors have determined a method of applying a hydrogelprotective layer onto a membrane biosensor in order to increase thestability of the membrane and in order to protect the membrane biosensorfrom interferents such as those present in blood.

Referring to the hydrogels, suitable polymers may either be regularhomopolymers containing substantially no other material in theirmatrices, slightly crosslinked homopolymers, or they may be copolymersprepared from two or more monomers.

Accordingly, in a fifth aspect the present invention consists in abiosensor for use in detecting the presence or absence of an analyte ina sample, the biosensor comprising an electrode and a bilayer membranecomprising a top layer and a bottom layer, the bottom layer beingproximal to and connected to the electrode such that a space existsbetween the membrane and the electrode, the conductance of the membranebeing dependent on the presence or absence of the analyte, the membranecomprising a closely packed array of amphiphilic molecules and aplurality of ionophores dispersed therein, and a layer of a hydrogelformed on top of the lipid bilayer membrane, the hydrogel allowing thepassage of the analyte molecule to be detected.

In a preferred form of the present invention, the hydrogel consists in athermosetting gel that is deposited onto the preformed lipid bilayermembrane at a temperature where the thermosetting gel is in the fluidphase and subsequently gels as the temperature is lowered below the gelssetting temperature, thus forming the protective gel membrane above thelipid bilayer membrane.

In a further preferred embodiment of the present invention, thethermosetting gel is an agar gel.

In a further preferred embodiment, the gel contains between 0.3-5% agar.

In a further preferred embodiment, the thermosetting gel is a gelatinegel.

In a further preferred embodiment of the present invention, the hydrogelconsists in an in situ polymerised hydrogel, where a solution of gelforming monomer is added to a preformed lipid bilayer membrane and issubsequently polymerised such that an hydrogel is formed as theprotective gel membrane above the lipid bilayer membrane.

In a further preferred embodiment, the gel forming monomer is an acrylicacid or an acrylic acid derivative that is polymerised by free radialpolymerisation.

In a further preferred embodiment, the hydrogel may be formed fromhydroxyalkyl acrylates and hydroxyalkyl methacrylates, for example,hydroxyethyl acrylate, hydroxypropyl acrylate andhydroxybutylmethacrylate; epoxy acrylates and epoxy methacrylates, suchas, for example, glycidyl methacrylate; amino alkyl acrylates and aminoalkyl methacrylates; N-vinyl compounds, such as, for example, N-vinylpyrrolidone; amino styrenes; polyvinyl alcohols and polyvinyl amines.

In a further preferred embodiment, the gel forming monomers consist ofacrylamide and a bisacrylamide cross-linker.

In a further preferred embodiment, the hydrogel is formed fromcross-linked hydroxyethyl acrylate or hydroxyethyl methacrylate or otherbiocompatible gel.

In a further preferred embodiment of the present invention, the hydrogelincorporates a number of groups that alter the partition of interferentsand/or analyte molecules into the gel layer by means of altering the netcharge of the hydrogel.

In a further preferred embodiment, the hydrogels contain enzymes such asurease that convert a substrate into an ionic species that is able to bedetected by the lipid bilayer membrane sensor. In the case of the enzymeurease, urea would be converted to ammonium ions which could be detectedby a lipid bilayer sensing membrane incorporating an ammonium selectiveionophore.

In an sixth aspect, the present invention consists in an electrodemembrane combination comprising an electrode and an ionically insulatingmonolayer membrane, the membrane comprising a closely packed array ofamphiphilic molecules and a plurality of ionophores dispersed therein,said amphiphilic molecules comprising within the same molecule ahydrophobic region, an attachment region attached to the electrode, ahydrophilic region intermediate said hydrophobic and attachment regions,the space formed by said hydrophilic region between the electrode andthe membrane being sufficient to allow the flux of ions through theionophores, and a head group attached to the hydrophobic portion of themolecule at a site remote from the hydrophilic region.

In a preferred embodiment of this aspect of the present invention, themonolayer membrane molecule has a hydrophobic region that consists ofoligomers of long chain amino acids, where the amino acids are linkedvia amide linkages, that are substituted at the nitrogen withhydrocarbon alkane groups. It is preferred that the structure of theamino acids is such that the amino group is typically separated from theacid group by an alkane chain of between 6-20 carbons long, and that thealkane chains attached to the nitrogen are typically between 10-20carbon atoms long and may be either saturated or contain unsaturatedgroups. Additionally, the alkane groups may consist of phytanyl orsimilar substituted alkane groups.

In a further preferred embodiment, the membrane consists in a monolayermembrane molecule where the hydrophobic region consists in a tertiary,trialkyl amine that is functionalised at two of the alkyl chains suchthat the tertiary amine is attached to a monoalkylsubstituted glycerol,monoalkyl substituted glutamic acid or other commonly used groupsnormally used in lipid synthesis in order to form dialkyl lipids. Theunfunctionalised alkyl group attached to the amino group may consists ofan hydrocarbon chain, typically of carbon chain length 1 to 20 carbonslong and may be unsaturated or additional alkyl substituted. The twofunctionalised alkyl chains attached to the amino group are typically 10to 20 carbon atoms long. The monoalkyl substituents of the monoalkylsubstituted glycerol, glutamic acid or other commonly used group wouldtypically be a hydrocarbon chain that is the same length as thefunctionalised alkyl chain attached to the amine group.

In a further preferred embodiment, the membrane consists in a monolayermembrane where the hydrophobic region consists in a tetra alkylatedpentaerythritol derivative where two of the alkyl chains arefunctionalised so as to allow attachment to monoalkyl substitutedglycerol, glutamic acid or other commonly used groups normally used inlipid synthesis. The unfunctionalised and functionalised alkyl groupsmay be the same as described above.

The head groups attached to the hydrophobic region of the membrane maycomprise of any of the hydrophilic head groups as described in the firstaspect of the present invention. Similarly, the attachment region andhydrophilic ionic reservoir region are any of the groups described inPCT/AU92/00132.

In yet a further preferred embodiment of the present invention theionophore is capable of transporting an ion that is produced by thereaction of an enzyme with its substrate, said enzyme being eithercovalently or non-covalently attached to the membrane surface.

Addition of a solution containing the substrate will cause the enzyme toproduce ionic species which will be transported by the ionophore acrossthe membrane, thus modulating the conductance properties of themembrane, which will be related to the amount of substrate present insolution.

In a preferred embodiment of the present invention, the enzyme is aurease producing ammonium ions from the substrate urea.

In the case where the enzyme is covalently attached to the membrane, itis preferred that the covalent attachment is via a proportion of thelipid molecules that are suitably functionalised for covalent attachmentto proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a structural diagram of a synthetic lipid for use in a bilayermembrane of the invention.

FIG. 2 is a structural diagram of a diol resulting from thedebenzylation of a bis-benzyl protected membrane spanning lipid.

FIG. 3 is a structural diagram of a membrane spanning lipid containingan alcohol head group.

FIG. 4 is a structural diagram of a membrane spanning lipid containing adicarboxy polyethylene glycol 400 head group.

FIG. 5 is a structural diagram of a further membrane spanning lipid ofthe present invention.

FIG. 6 is a graph of the impedance spectra comparing sealed and unsealedmembranes.

FIG. 7 is a structural diagram for reservoir phytanyl lipid.

FIG. 8 is a structural diagram for a homologue of the reservoir phytanyllipid of FIG. 7.

FIG. 9 is a graph of the impedance spectra of electrodes in the presenceand absence of streptavidin solution and gramicidin derivative.

FIG. 10 is a series of traces of impedance spectra for bilayer membraneswith varying concentrations of GMO, tetradecane and gramicidin-biotinconjugate conjugate in the layers.

FIG. 11 is a second series of traces of impedance spectra for bilayermembranes with varying concentrations of GMO, tetradecane andgramicidin-biotin conjugate in the layers.

FIG. 12 is a third series of traces of impedance spectra for bilayermembranes in which the concentration of gramicidin in the bottom layeris varied.

FIG. 13 is a structural diagram for liker gramicidin B, a functionalizedgramicidin comprising a gramicidin backbone, a hydrophilic group and adisulfide group.

DETAILED DESCRIPTION OF THE INVENTION

In order that the nature of the present invention may be more clearlyunderstood preferred forms thereof will now be described with referenceto the following examples.

EXAMPLE 1

Synthesis of membrane spanning lipids

1,3-Benzylidine glycerol was prepared according to the method of H. S.Hill et al in Carbohydrates and Polysaccharides, 50, (1928), 2242-2244.The 1,3-benzylidine glycerol was then treated with sodium hydride intetrahydrofuran and phytanyl bromide under reflux for 24 hours to givethe glycerol 2-phytanylether 1,3-benzylidine. This ether was thentreated with a mixture of potassium borohydride and boron trifluorideetherate in refluxing tetrahydrofuran for 24 hours to give the glycerol1-benzylether 2-phytanylether. The product was then treated with sodiumhydride and 1,16-dibromohexadecane in refluxing tetrahydrofuran for 24hours to yield the glycerol 1-benzylether 2-phytanylether3-(16-bromohexadecyl) ether. The homologous compounds using1,12-dibromododecane, or 1,14-dibromotetradecane were produced insimilar fashion. Treatment of the product with biphenol and sodiumhydride in refluxing tetrahydrofuran gave the bis-benzyl protectedmembrane spanning lipid which, after isolation, was debenzylation usingpalladium on charcoal to give the diol shown in FIG. 2. Addition of areservoir component as described in PCT/AU92/00132 in the presence ofdicyclohexylcarbodiimide and dimethylamino pyridine gave the membranespanning lipid shown in FIG. 3 which contains an alcohol head group(MSL-OH). Treatment of this membrane spanning lipid with thediacidchloride of an acid functionalised polyethylene glycol 400(average molecular weight 400 g/mol) followed by an aqueous workup gavethe membrane spanning lipid shown in FIG. 4 (MSLPEG400COOH) whichcontains a dicarboxy polyethylene glycol 400 head group. Treatment ofthe diol shown in FIG. 2 with firstly, one equivalent ofBOC-glycine/dicyclohexylcarbodiimide and dimethylamino pyridine andisolation of the monosubstituted compound, secondly with trifluoroaceticacid to remove the BOC group, thirdly treatment with abiotin-xx-N-hydroxysuccinimide (where the X group is an 6-aminocaproicacid group), and fourthly, treatment of the product with the reservoircomponent as above in the presence of dicyclohexylcarbodiimide anddimethylamino pyridine gave the membrane spanning lipid as shown in FIG.5 (MSLXXB).

EXAMPLE 2

Effect of Small Spacer Compound on Conduction through the First Layer

A freshly prepared evaporated 2 mm² gold on glass electrode was immersedin a solution of linker (a) and bis(2-hydroxyethyl)disulfide (HEDS) atvarious ratios (final concentration was 0.2 mM in ethanol), within fiveminutes of preparation. After allowing the disulfide species to adsorbfor a period of between 30 minutes to 3 days the electrodes were rinsedwith ethanol, dried and clamped in a containment vessel. Two microlitresof an ethanol solution of glycerol monooleate (GMO) (140 mM) andvalinomycin (GMO/valinomycin ratio 3000:1) with 8% tetradecane (v/v) wasadded to the electrode. The electrode was then rinsed twice with 0.5 mlof 0.1M saline solution. After the impedance spectrum was obtained, thesodium chloride solution was exchanged with 0.1M potassium chloridesolution. The absolute impedance values at 1 Hz are shown in Table 1below.

                  TABLE 1                                                         ______________________________________                                        Ratio Linker            KCl(0.1M)                                             (A):HEDS      NaCl(0.1M)                                                                              log/Z/ at 1Hz                                         ______________________________________                                        1:0           7.62      7.40                                                  20:1          7.50      7.25                                                  5:1           7.85      6.81                                                  1:1           7.62      6.61                                                  1:2           7.81      6.35                                                   1:20         7.54      6.38                                                  ______________________________________                                    

As can be clearly seen, the conduction of the potassium via thevalinomycin increases as the linker (A) molecule is spaced further apartby the HEDS molecule.

When the above experiment is repeated using the membrane spanning lipidshown in FIG. 3 (MSL-OH) with various HEDS ratios similar results wereobtained as shown in Table 2. At high MSL-OH ratios the lipid membranesystems appear to contain multilammellar structures, hence the overallhigh impedance

                  TABLE 2                                                         ______________________________________                                                                  KCl(0.1M)                                           Ratio MSL-OH:HEDS                                                                             NaCl(0.1M)                                                                              log/Z/ at 1Hz                                       ______________________________________                                        1:0             7.8       7.3                                                  1:10           7.5       6.4                                                  1:100          7.0       6.1                                                 0:1             6.9       6.1                                                 ______________________________________                                    

Adsorpotion of a monolayer of MSLPEG400COOH onto a 2 mm² gold electrodewith no HEDS, followed by the addition of GMO/tetradecane as describedabove, resulted in a bilayer membrane with an impedance of 200 kohms at100 Hz. Conversely, an electrode with a MSLOH first layer followed bythe addition of GMO/tetradecane as described above resulted in amembrane containing thicker or multilamellar structures as seen by thehigh impedance of 650 kohms at 100 Hz.

EXAMPLE 3

Formation of a lipid bilayer membrane without a sealing alkane

A freshly prepared evaporated 2 mm² gold on glass electrode was immersedin a solution of linker (A) and bis(2-hydroxyethyl)disulfide (HEDS) at aratio of 8:2 (final concentration was 0.2 mM in ethanol), within fiveminutes of preparation. After allowing the disulfide species to adsorbfor a period of between 30 minutes to 3 days the electrodes were rinsedwith ethanol, dried and clamped in a containment vessel. Two microlitresof an ethanol solution of glycerol monooleate (GMO) (140 mM) ormono-11-eicosenoin (140 mM) or glycerol 1-phytanyl ether (140 mM) wasadded to the electrode. These solutions contained no alkane co-solvent.The electrode was then rinsed twice with 0.5 ml of 0.1M saline solutionand impedance spectra were obtained and are shown in FIG. 6. It wasfound that the glycerol 1-phytanyl ether formed useable sealed bilayermembranes whereas both the GMO and the mono-11-ecosenoin did not formsealed membranes.

EXAMPLE 4

Synthesis and Formation of Bilayer Membranes using a Reservoir PhytanylLipid

Reservoir phytanyl lipid (B) is shown in FIG. 7. This compound wassynthesised from4,18,21-trioxo-36-phenyl-34,35-dithio-5,8,11,14,17,22,25,28,31-nonaoxohexatricontanoicacid and phytanol in the presence of dicyclohexylcarbodiimide anddimethylamino pyridine. The homologous reservoir phytanyl lipid (C)shown in FIG. 8 was synthesised in analogous fashion from the suitablehydrophilic precursor and phytanol.

A bilayer membrane was formed onto a freshly evaporated gold electrodeusing the protocol described in Example 2 but in the absence of anysmall spacer molecule. Thus a solution of reservoir phytanyl lipid (B)or (C) in ethanol was contacted with the gold electrode surface,followed by rinsing of the electrode.

A bilayer membrane was then formed by addition of 5 microlitres of anethanol solution containing glycerol 1-phytanyl ether (140 mM) andvalinomycin (glycerol 1-phytanyl ether/valinomycin 1500:1) followed by0.1M sodium chloride solution. Impedance spectra were taken before andafter addition of potassium chloride solution and values at 10 Hz areshown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Reservoir                KCl(0.1M)                                            Phytanyl Lipid NaCl(0.1M)                                                                              log/Z/ at 10Hz                                       ______________________________________                                        1:0            7.8       7.3                                                   1:10          7.5       6.4                                                   1:100         7.0       6.1                                                  0:1            6.9       6.1                                                  ______________________________________                                    

EXAMPLE 5

Reduced effect of serum on lipid bilayers by incorporation of lipidscontaining PEG 2000 head groups

A freshly prepared evaporated 2 mm² gold on glass electrode was immersedin a solution of linker (A) and bis(2-hydroxyethyl)disulfide (HEDS) atan 8:2 ratio (final concentration was 0.2 mM in ethanol), within five 25minutes of preparation. After allowing the disulfide species to adsorbfor a period of between 30 minutes to 3 days the electrodes were rinsedwith ethanol, dried and clamped in a containment vessel. Two microlitresof an ethanol solution of glycerol monooleate (GMO) (140 mM), succinicacid phytanol half-ester PEG2000 half-ester (PSP-2000) (1-4 mol%relative to GMO) and gramicidin (GMO/gramicidin ratio 1000:1) with 8%tetradecane (v/v relative to ethanol) was added to the electrode. Theelectrode was then rinsed twice with 0.5 ml of 0.1M saline solution.After the impedance spectrum was obtained, 2 microlitres of whole plasmawas added and the impedance spectrum again measured. The absoluteimpedance values of 1 Hz are shown in Table 4 for various GMO/PSP-2000lipid ratios.

                  TABLE 4                                                         ______________________________________                                        Ratio GMO/PSP-           KCl(0.1M)                                            2000           NaCl(0.1M)                                                                              log/Z/ at 1Hz                                        ______________________________________                                        100:0          7.3       6.5                                                  99:1           7.1       6.7                                                  98.2           7.1       7.0                                                  97:3           6.9       6.8                                                  96:4           6.9       6.4                                                  95:5           7.0       6.4                                                  ______________________________________                                    

As can be seen the effect of plasma on the membranes is most effectivelyreduced at ratios of 1-3 mol % of the PSP-2000 lipid.

EXAMPLE 6

Formation of a protective hydrogel onto a lipid membrane

A lipid membrane was produced onto a gold electrode using the protocoldescribed in Example 2. Excess saline was removed from the containmentvessel and ten microlitres of a solution of agar (0.5-5% w/v) in 0.1Msodium chloride was added to the lipid membrane assembly at 40° C. Themembrane assembly was allowed to cool to room temperature whereupon theagar gelled forming a protective membrane over the intact lipidmembrane. In the case where the lipid membrane contained valinomycin asthe ionophore, addition of a potassium solution caused a decrease in theimpedance as expected, although the response times wereslower--approximately 15 seconds compared to less than 1 second withoutthe gel membrane. It was also found that addition of whole plasma orserum did not have any effect on the lipid membrane for at least 20minutes when a 0.3% w/v agar gel was used or 1.5 hours when a 3% w/vagar gel was used.

A hydrogel could be also formed onto a lipid membrane by addition of tenmicrolitres of a solution of acrylamide (4% w/v) andN'N'-bis-methylene-acrylamide (0.3% w/v) in 0.1M sodium chloridetetramethylethylene diamine (0.01%), followed by addition of twomicrolitres of a 10% solution of ammonium persulfate in 0.1M sodiumchloride solution. The acrylamide gelled giving an intact lipid membraneelectrode combination which, when the lipid contained the valinomycinionophore responded to potassium in the usual manner and which protectedthe lipid membrane from non-specific effects of serum and plasma for upto one hour.

EXAMPLE 7

Formation of an enzyme/ion selective electrode combination electrode

A lipid membrane was formed according to the protocol as described inExample 2 but where the ionophore is nonactin at a GMO/nonactin ratio of3000:1. To the electrode membrane combination was added two microlitresof a 0.5 mg/ml solution of urease in 0.1M sodium chloride solution,allowing the urease to non-specifically bind to the lipid membranesurface as monitored by impedance spectroscopy, as a control identicalelectrodes were formed but without the urease addition. After 10 minutes10 microlitres of a solution of urea (0.1M in 0.1M sodium chloridesolution) was added to the urease/ion selective electrode combinationand to the control. It was found that on addition of the urea theimpedance of the urease/ion selective electrode dropped substantiallymore (impedance at 1 Hz dropped from log 7.3 ohms to log 7.1 ohms) thanthat of the control (impedance at 1 Hz dropped from log 7.3 ohms to log7.25 ohms). It is expected that the urease converts the urea to ammoniumwhich is transported by the nonactin across the lipid membrane. A majoradvantage of this enzyme/ion selective electrode over conventionalenzyme/ion selective electrodes is that it is possible to produceinexpensive, single use sensors with fast response times.

EXAMPLE 8

Method of adsorbing Gramicidin B

1st layer

Onto freshly prepared 2 mm² gold electrodes was deposited 2 μl of aethanolic solution containing 140 mM glycerol monoleate, 140 μMreservoir lipid A, 14 μM MSLXXB, 1.4 μM Gramicidin B. 100 μl 0.1 μM NaClwas immediately added and the assembly allowed to stand overnight. Thesaline solution was then removed, the assembly rinsed with ethanol(5×100 μl) and drained.

2nd layer

To the above prepared electrode was added 5 μl of a ethanolic solutionof 140 mM glycerol monooleate and 1.4 μM biotin-gramicidin conjugate, 2%(v/v) tetradecane. The assembly was immediately treated with 100 μl 0.1MNaCl. The saline solution is removed and replaced with fresh saline (100μl) five times.

FIG. 9 shows the impedance of the electrodes before (a), and after (b)challenge with 1 μl 0.05 mg/ml streptavidin solution (0.1M NaCl). Theimpedance trace obtained for the sealed membrane, i.e. withoutgramicidin derivative in the 2nd layer, is shown in (c).

Conducting membranes that respond to the addition of streptavidin canalso be obtained by varying the method described above with thefollowing:

1) type of membrane spanning lipid added

2) replacing glycerol monooleate with other different chain lengthderivatives or glycerol monooleate ether derivatives

3) the concentration of MSLXXB from 1 μM to 140 mM

4) the concentration of Gramicidin B from 1 μM to 14 μM

5) replacing reservoir phytanyl lipid B with reservoir lipid A orreservoir phytanyl lipid C in the concentration range 10 μM to 1 mM.

6) saline can be omitted from the first layer, and glycerol monooleatecan also be omitted from the first layer

7) ethanol can be replaced with other polar solvents such as methanol ordioxane

8) the 2nd layer can be made up with or without addition of alkanes suchas tetradecane.

EXAMPLE 9

First Layer

Onto a freshly prepared (by evaporation or sputtering) 2 mm² goldelectrode is placed 2 ml of a solution comprising glycerolmonooleate(0.14M), reservoir lipid A (1.4 mM) and linker gramicidin B (0.014 mM)in a 98:1 (v/v) mixture of ethanol and tetradecane. The electrode/wellassembly is then immediately treated with 100 ml of 0.1M NaCl and theassembly is allowed to stand overnight. The saline solution is thenremoved and the assembly is washed (5×100 ml ethanol) and drained.

Second Layer

To the above prepared electrode is added a solution of gramicidin-biotinconjugate(0.14 mM) and glyceryl monooleate(0.14M) in ethanol(5 ml). Theassembly is then immediately treated with 0.1M saline(100 ml). Thesaline solution is the removed and replaced with fresh saline(100 ml)five times.

Membranes were formed as described in the above example but with varyingconcentrations of gramicidin in the two layers. The impedance of themembranes was measured and the membranes challenged with 1 ml 0.5 mg/mlstreptavidin. The impedance traces obtained are shown in FIGS. 10-12.

In each of the traces shown in FIGS. 10(a-d) the bottom layer consistedof 0.14 mM double length reservoir gramicidin, 1.4 mM GUDRUN, 140 mMglyceryl monooleate (GMO), 10% tetradecane. The top layers eachconsisted of GMO, tetradecane (10%) and varying concentrations ofgramicidin-biotin conjugate: FIG. 10a-0; FIG. 10b-0.0014 mM; FIG.10c-0.014 mM; FIG. 10d-0.14 mM.

In FIG. 10e only 0.14 mM gramicidin-biotin cojugate, 140 mM GMO,tetradecane 10% solution was applied to a fresh gold electrode.

FIG. 11 is the same as FIG. 10 except that the concentration of doublelength reservoir gramicidin in the bottom layer for traces a-d was 0.014mM. In FIG. 11e only 0.014 mM gramicidin-biotin cojugate, 140 mM GMO,tetradecane 10% solution was applied to a fresh gold electrode.

In FIG. 12 the concentration gramicidin-biotin conjugate in the toplayer was maintained constant at 0.14 mM and the concentration of doublelength reservoir gramicidin in the bottom layer varied. FIG. 12a-1.4 nM;FIG. 12b-14 nM; FIG. 12c-140 nM; FIG. 12d-1.4 mM; FIG. 12e-14 mM; FIG.12f-140 mM. FIG. 10e is repeated as FIG. 12g for comparison.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

We claim:
 1. A method of producing an electrode membrane combination,the method comprising the following sequential steps:(1) Forming asolution comprising ion channels having attached at an end thereof areservoir region, the reservoir region including a hydrophilic group andan attachment group; and a reservoir lipid, the reservoir lipidcomprising a hydrophobic region, a hydrophilic region and an attachmentregion in a polar carrier solvent; (2) Contacting the electrode with thesolution from step (1), the composition of the electrode and theattachment groups and the attachment regions being selected such thatthe attachment groups and the attachment regions chemisorb to theelectrode; (3) After a period of incubation rinsing the coated electrodefrom step (2) to remove unbound material; (4) Adding to the rinsedelectrode from step (3) a solution comprising ion channels and a lipidin a carrier solvent; and (5) Adding to the electrode from step (4) anaqueous solution such that a lipid bilayer membrane coating theelectrode is formed.
 2. A method as claimed in claim 1 in which thesolution in step 1 further includes a lipid.
 3. A method as claimed inclaim 1 in which the ion channels are gramicidin or analogues orderivatives thereof.
 4. A method as claimed in claim 1 in which the ionchannels are linker gramicidin B.
 5. A method as claimed in claim 1 inwhich the reservoir lipid is23-(20'-Oxo-19'-oxaeicosa-(Z)-9'-ene)-70-phenyl-20,25,28,42,45-pentaoxo-24-aza-19,29,32,35,38,41,46,47,52,55-decaoxa-58,59-dithiahexaconta-(Z)-9-ene or reservoir phytanyl lipid (B) orreservoir phytanyl lipid (C).
 6. A method as claimed in claim 1 in whichthe reservoir lipid used in the solution in step 1 has a biotincontaining head group.
 7. A method as claimed in claim 1 in which thereservoir lipid used in step 1 has a head group used to couple thereservoir lipid to a protein molecule.
 8. A method as claimed in claim 1in which the reservoir lipid used in step 1 has as a head group apolyethylene glycol or phosphatidyl choline group.
 9. A method asclaimed in claim 1 in which the lipid in the solution in step 1 and/orstep 4 is glycerol monoalkenoate.
 10. A method as claimed in claim 9 inwhich the lipid is a glycerol monoalkenoate where the alkenoate groupmay be an unsaturated hydrocarbon chain of between 16-22 carbons inlength.
 11. A method as claimed in claim 1 in which the lipid in thesolution in step 1 and/or step 4 is glycerol monooleate.
 12. A method asclaimed in claim 1 in which the lipid in step 4 is a glycerol monoalkylether where the alkyl group is a hydrocarbon chain of between 16-22carbons in length.
 13. A method as claimed in claim 1 in which the lipidin step 4 is glycerol monooleate, glycerol monopalmitoleic,mono-11-eicosenoin, or mono-erucin.
 14. A method as claimed in claim 1in which the lipid in step 4 is glycerol monophytanyl ether.
 15. Amethod as claimed in claim 1 in which the lipid in step 4 is a mixtureof glycerol monoalkyl ether or glycerol monoalkenoate and a lipid wherethe head group is a polyethylene glycol group of between 600-6000 g/mol.16. A method as claimed in claim 1 in which the lipid in step 4 is amixture of glycerol monoalkyl ether or glycerol monoalkenoate and 1-3%of a lipid where the head group is a polyethylene glycol group.
 17. Amethod as claimed in claim 16 in which the polyethylene glycol has amolecular weight in the range of 600-3000 g/mol.
 18. A method as claimedin claim 16 in which the polyethylene glycol containing lipid comprisesin the same molecule a phytanyl group attached to a succinate group atone end and a polyethylene glycol 2000 attached to the other end of thesuccinate.
 19. A method as claimed in claim 1 in which the lipid in step4 is a mixture of glycerol monoalkyl ether or glycerol monoalkenoate anda lipid where the head group is a phosphatidyl choline head group.
 20. Amethod as claimed in claim 1 in which the lipid in step 4 is a mixtureof glycerol monoalkyl ether or glycerol monoalkenoate and up to 20% of alipid where the head group is a phosphatidyl choline head group.
 21. Amethod as claimed in claim 1 in which the lipid in step 4 is a mixtureof glycerol monoalkyl ether or glycerol monoalkenoate and up to 20% of alipid where the head group is a phosphatidyl choline head group and upto 3% of a lipid where the head group is a polyethylene glycol ofmolecular weight between 600-3000 g/mol.
 22. A method as claimed inclaim 1 in which the lipid in step 4 is a mixture of glycerol monoalkylether or monoalkenoate and a lipid where the head group is aGalβ1-3-GalNAcβ1-4Gal(3-2α-NeuAc)β1-4Glc-1-1 ceramide carbohydrate headgroup.
 23. A method as claimed in claim 1 in which the solutions insteps (1) and (4) contain less than 2% decane, dodecane, tertradecane orhexadecane.
 24. A method as claimed in claim 1 in which the solutions insteps (1) and (4) contain no decane, dodecane, tetradecane orhexadecane.